Luminescence detection workstation

ABSTRACT

A luminescence detecting apparatus and method for analyzing luminescent samples is disclosed. Luminescent samples are placed in a plurality of sample wells in a tray, and the tray is placed in a visible-light impervious chamber containing a charge coupled device camera. The samples may be injected in the wells, and the samples may be injected with buffers and reagents, by an injector. In the chamber, light from the luminescent samples pass through a collimator, a Fresnel field lens, a filter, and a camera lens, whereupon a focused image is created by the optics on the charge-coupled device (CCD) camera. The use of a Fresnel field lens, in combination with a collimator and filter, reduces crosstalk between samples below the level attainable by the prior art. Preferred embodiments of the luminescence detecting apparatus and method disclosed include central processing control of all operations, multiple wavelength filter wheel, and robot handling of samples and reagents. Preferred embodiments of processing software integrated with the invention include elements for mechanical alignment, outlier shaving, masking, manipulation of multiple integration times to expand the dynamic range, crosstalk correction, dark subtraction interpolation and drift correction, multi-component analysis applications specifically tailored for luminescence, and uniformity correction.

This application claims the benefit from Provisional Application SerialNo. 60/144,891, filed Jul. 21, 1999. The entirety of that provisionalapplication is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of apparatus and methods fordetecting and quantifying light, emissions, and more particularly, todetecting and quantifying light emitted from luminescent-based assays.Still more particularly, this invention pertains to apparatus andmethods for detecting and quantifying luminescence such asbioluminescence and/or chemiluminescence from luminescent assays as anindicator of the presence or amount of a target compound. Preferredembodiments of the invention include as an imaging device a chargecoupled device (CCD) camera and a computer for analyzing data collectedby the imaging device. Further preferred embodiments include thecapacity for use in high throughput screening (HTS) applications, andprovide for robot handling of assay plates.

2. Description of the Related Art

The analysis of the luminescence of a substance, and specifically theanalysis of either bioluminescence (BL) or chemiluminescence (CL), isbecoming an increasingly useful method of making quantitativedeterminations of a variety of luminescent analytes.

Recently, methods have been introduced that utilize luminescencedetection for quantitatively analyzing analytes in an immunoassayprotocol. Such luminescence immunoassays (LIA) offer the potential ofcombining the reaction specificity of immunospecific antibodies orhybridizing nucleic acid sequences and similar specific ligands with thehigh sensitivity available through light detection. Traditionally,radioactive reagents have been used for such purposes, and thespecificity and sensitivity of LIA reagents is generally comparable tothose employing traditional radiolabelling. However, LIA is thepreferred analytical method for many applications, owing to the nontoxicnature of LIA reagents and the longer shelf lives of LIA reagentsrelative to radioactive reagents.

Among other luminescent reagents, chemiluminescent compounds such as1,2-dioxetanes, developed by Tropix, Inc. and other stablechemiluminescent molecules, such as xanthan esters and the like, are incommercial use. These compounds are triggered to release light throughdecomposition triggered by an agent, frequently an enzyme such asalkaline phosphatase, which is present only in the presence, or specificabsence, of the target compound. The detection of light emission is aqualitative indication, and the amount of light emitted can bequantified as an indicator of the amount of triggering agent, andtherefore target compound, present. Other well known luminescentcompounds can be used as well.

Luminescent release may sometimes be enhanced by the presence of anenhancement agent that amplifies or increases the amount of lightreleased. This can be achieved by using agents which sequester theluminescent reagents in a microenviroment which reduces suppression oflight emission. Much biological work is done, perforce, in aqueousmedia. Water typically suppresses light emission. By providingcompounds, such as water soluble polymeric onium salts (ammonium,phosphonium, sulfonium, etc.) small regions where water is excluded thatmay sequester the light emitting compound may be provided.

The majority of instrumentation used to monitor light emitting reactions(luminometers) use one or more photomultiplier tubes (PMTs) to detectthe photons emitted. These are designed to detect light at the low lightlevels associated with luminescent reactions. The rate at which a PMTbased microplate luminometer can measure signal from all wells of theplate is limited by the number of PMTs used. Most microplateluminometers have only one PMT so a 384 well plate requires four timeslonger than is required to read a 96-well plate.

The nature of biological research dictates that numerous samples beassayed concurrently, e.g., for reaction of a chemiluminescent substratewith an enzyme. This is particularly true in gene screening and drugdiscovery, where thousands of samples varying by concentration,composition, media, etc. must be tested. This requires that multiplesamples be reacted simultaneously, and screened for luminescence.However, there is a need for high speed processing, as thechemiluminescence or bioluminescence may diminish with time.Simultaneously screening multiple samples results in improved datacollection times, which subsequently permits faster data analysis, andcontingent improved reliability of the analyzed data.

In order for each specific sample analyte's luminescence to be analyzedwith the desired degree of accuracy, the light emission from each samplemust be isolated from the samples being analyzed concurrently. In suchcircumstances, stray light from external light sources or adjacentsamples, even when those light levels are low, can be problematic.Conventional assays, particularly those employing high throughputscreening (HTS) use microplates, plastic trays provided with multiplewells, as separate reaction chambers to accommodate the many samples tobe tested. Plates currently in use include 96- and 384-well plates. Inresponse to the increasing demand for HTS speed and miniaturization,plates having 1,536 wells are being introduced. An especially difficultimpediment to accurate luminescence analysis is the inadvertentdetection of light in sample wells adjacent to wells with high signalintensity. This phenomenon of light measurement interference by adjacentsamples is termed ‘crosstalk’ and can lead to assignment of erroneousvalues to samples in the adjacent wells if the signal in those wells isactually weak.

Some previously proposed luminometers include those described in U.S.Pat. No. 4,772,453; U.S. Pat. No. 4,366,118; and European Patent No. EP0025350. U.S. Pat. No. 4,772,453 describes a luminometer having a fixedphotodetector positioned above a platform carrying a plurality of samplecells. Each cell is positioned in turn under an aperture through whichlight from the sample is directed to the photodetector. U.S. Pat. No.4,366,118 describes a luminometer in which light emitted from a lineararray of samples is detected laterally instead of above the sample.Finally, EP 0025350 describes a luminometer in which light emittedthrough the bottom of a sample well is detected by a movablephotodetector array positioned underneath the wells.

Further refinements of luminometers have been proposed in which a liquidinjection system for initiating the luminescence reaction just prior todetection is employed, as disclosed in EP 0025350. Also, a temperaturecontrol mechanism has been proposed for use in a luminometer in U.S.Pat. No. 4,099,920. Control of the temperature of luminescent samplesmay be important, for example, when it is desired to incubate thesamples at an elevated temperature.

A variety of light detection systems for HTS applications are availablein the market. These include the LEADseeker™ from Amersham/Pharmacia,the ViewLux™ offered by PerkinElmer and CLIPR™ from Molecular Devices.These devices are all expensive, large dimensioned (floorbased models),exhibit only limited compatibility with robotic devices for platepreparation and loading, have a limited dynamic range, and/or useoptical detection methods which do not reduce, or account for,crosstalk. The optical systems used are typically complex teleconcentricglass lens systems, which may provide a distorted view of wells at theedges of the plates, and the systems are frequently expensive, costingin excess of $200,000.00. Perhaps the most popular detection apparatusis the TopCount™, a PMT-based detection system from Packard. Althoughthe TopCount™ device has a desirable dynamic range, it is not capable ofreading 1,536 well plates, and it does not image the whole platesimultaneously.

Crosstalk from adjacent samples remains a significant obstacle to thedevelopment of improved luminescence analysis in imaging-based systems.This can be appreciated as a phenomenon of simple optics, whereluminescent samples produce stray light which can interfere with thelight from adjacent samples. Furthermore, the development ofluminometers capable of detecting and analyzing samples with extremelylow light levels are particularly vulnerable to crosstalk interference.

SUMMARY OF THE INVENTION

In order to meet the above-identified needs that are unsatisfied by theprior art, it is a principal object and purpose of the present inventionto provide a luminescence detecting apparatus that will permit theanalysis of luminescent samples. It is a further object of the presentinvention to provide a luminescence detecting apparatus capable ofsimultaneously analyzing a large number of luminescent samples. In apreferred embodiment of the present invention, a luminescence detectingapparatus is provided that simultaneously analyzes multiple samples heldin wells, where the well plates contain as many as 1,536 wells. Thepresent invention further includes robot handling of the multiple welltrays during analysis.

It is yet another object of the present invention to provide aluminescence detecting apparatus capable of analyzing low light levelluminescent samples, while minimizing crosstalk from adjacent samples,including and especially minimizing crosstalk from adjacent samples withhigher light level output than the sample to be analyzed.

The apparatus of this invention employs a Fresnel lens arrangement, witha vertical collimator above the well plate, with dimensions to match thenumber of wells. Thus, a 1,536-well plate will employ a dark collimatorabove the plate with 1,536 cells in registry with the wells of theplate. Fixed above the collimator is a Fresnel lens, which refracts thelight such that the view above the lens appears to be looking straightdown into each well, regardless of its position on the plate, even atthe edges.

Above the Fresnel lens is a CCD camera arranged so as to take the imageof the entire plate at one time, viewing through a 35 mm wide anglelens, to give whole plate imaging on a rapid basis. Between the CCD andFresnel/collimator is a filter, typically arrayed on a filter wheel,disposed at an angle to the lens. The filter is selected to permit thepassage of the specific wavelength of the light emitted, and reflect orabsorb all others. Several filters may be provided on the wheel, topermit sequential detection of light emitted from multiple reagentsemitting light at different wavelengths.

The samples are fed to the optical detection platform through a loadingdevice designed to work well with robotic and automated preparationsystems. The well-plate, with reaction mixture already provided, isplaced on a shuttle by a human, or preferably, robot. Alignment of theplate on the shuttle may be relatively coarse, notwithstanding therequirement for tight tolerances to match the collimator grid array. Asthe shuttle leaves the loading position, a resilient means urges theplate into strict conformal alignment. The shuttle positions the plateunder an overhead injection bar, which may accommodate up to sixteenwells in a column at one time. If not previously added, a triggeringagent or luminescent reagent is added to the sample wells, and the plateindexes forward to load the next column of wells across the plate. Theshuttle then advances through a door into the sample chamber, and theplate is aligned with the collimator and the Fresnel lens. Since manyreactions proceed better, or only, at elevated temperatures, the samplechamber is insulated, and provided with heating means, for heating theair in or provided to the chamber. In order to maintain temperature inthe chamber close to room temperature and to accurately controltemperature, the chamber may also be provided with a heat exchanger.

The light emission from the entire multiple well plate is imaged atonce, with subsequent imaging through a different filter if multiplewavelengths are employed. The signal obtained is processed to furtherreduce crosstalk reduced by the collimator and the presence and amountof luminescence is quickly detected and calculated by a personalcomputer using automated software. Data is then reported as intensityper well or further analyzed relative to specific assay standards.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 is a cross section of a preferred embodiment of a luminescencedetecting apparatus according to the present invention;

FIG. 2 is a detailed cross section of the optics of a luminescencedetecting apparatus according to the present invention.

FIG. 3 is a cross-sectional view of the plate transport system of theinvention.

FIG. 4 is a perspective illustration of the injector arm assembly of theinvention.

FIG. 5 is an exploded view of the filter wheel assembly.

FIG. 6 is a cross-sectional view of the optical housing.

FIG. 6A is a plan view of a robotic mechanism of the invention.

FIG. 7 is a flow chart illustration of the processing method of theinvention.

FIGS. 8-15 are illustrations of the results obtained using the inventionin Examples 1-10, respectively.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring now to the drawings, wherein like reference numerals designateidentical or corresponding parts throughout the several views, and moreparticularly to FIG. 1 thereof, a preferred embodiment of theluminescence detecting apparatus of the present invention uses a shuttleor tray to carry a micro plate (plate) 10 comprising a plurality ofsample wells 20 which may in the preferred embodiment number as many as1,536 or more. Persons of ordinary skill in the relevant art willrecognize that the number of sample wells 20 is limited only by thephysical dimensions and optical characteristics of the luminometerelements, and not by the technology of the present invention. The samplewells 20 may be filled with analyte manually, or robotically, prior todelivery to the inventive apparatus. Agents necessary forchemiluminescence may be filled automatically via the injector 30, towhich analyte is supplied through an array of supply tubes 40 or priorto placing the plate on the tray. Typicially, the sample wells willcontain chemiluminescent reagents. These reagents emit light atintensities proportional to the concentration of analyte in the sample.This light can be very low intensity and requires an instrument withsufficient sensitivity to achieve the desired detection limits.

The operation of injector 30 is controlled by central processor 50which, in the preferred embodiment, may control the operation of allelements of the luminometer of the present invention. Data collection,analysis and presentation may also be controlled by the processor 50.Further in a preferred embodiment of the present invention, the injector30 may also be used to add buffer solutions to the analytes and also toadd reagents that enable “glow” and/or “flash” luminescence imaging,that is sustained or brief, intense emission, respectively, all undercontrol of central processor 50.

After the analytes are placed in the sample wells 20, plate 10 is placedin sample chamber 55, which is located in optical chamber 60 at a fixedfocal distance from and directly under the charge-coupled device (CCD)camera 70, in order to permit the CCD camera to image the luminescentsample accurately. The sample chamber 55 is preferably capable ofprecise temperature control, as many luminescent reagents and specificluminescent reactions are temperature dependent. Temperature control isprovided by central processor 50, which can vary the temperature foreach individual sample plate 10, as central processor 50 controls themovement and injection of the sample wells 20 in each sample tray 10. Ina preferred embodiment of the luminometer of the present invention,central processor 50 also controls an industrial robot (not shown) whichperforms the activities involving analyte handling in the luminometer ofthe present invention.

With the plate 10 placed in the sample chamber 55, the optics 80 deliverthe image of the complete microplate 10 as a single image to the CCDcamera 70.

Although the operation of the luminometer of this invention is anintegral, continuous practice, and all elements of the luminometercooperate together to provide precise, accurate and reliable data, theinvention may be more easily understood by reference to three separate,integrated systems, the optics system, the mechanical system and theprocessing system. Each is discussed in turn, with a discussion ofexamples of the operation as a whole to follow.

Optics System

Turning now to FIG. 2, the optics 80 are shown in further detail.Luminescent emission 100 from the analyte in plate well 20 located inthe plate 10 travels first through dark collimator 110, which permitsonly parallel and semi-parallel light rays to exit the sample wells 20for eventual imaging by the CCD camera 70. The effect of collimationassists with the prevention of stray light from the sample wells 20 andwith the elimination of crosstalk between luminescent samples. Thecollimator 110 may be sealably engaged, or in close proximity, to thesample tray 10, to enhance the restriction of stray light from thesamples. Each well 10 is in strict registration and alignment with acorresponding grid opening in collimator 110. From the collimator 110,the luminescent radiation passes through a Fresnel field lens 120, whichfocuses the light toward camera lens 140. In a preferred embodiment ofthe present invention, the collimator 110 and Fresnel field lens 120 arepackaged in a cassette that can be changed by the user. Such anequipment change may be necessitated by varying optical characteristicsof different analytes and different well distributions in plates.

The use of a Fresnel field lens is preferable to alternative opticaldevices for several reasons. Initially, improvements in design andmaterials have capitalized on the superior optical capabilities of theFresnel lens, while virtually eliminating its once inherent limitations.Today, many Fresnel lenses are made of molded plastic, creating analmost flawless surface with very little scatter light. The eliminationof scatter light is an important element of eliminating crosstalkbetween adjacent samples in the luminometer of the present invention.Furthermore, improved types of plastics commonly employed in themanufacturing of Fresnel lenses and other optical devices have opticalqualities equivalent to ground glass lenses.

Using high tech processes such as computer-controlled diamond turning,complex aspheric surfaces can be cut into a long lasting mold forcasting Fresnel lenses. In this manner, Fresnel lenses can bemanufactured to produce the precise optical imaging effect that is mostefficient for a charge coupled device camera, as in the presentinvention. Also, Fresnel lenses offer an advantage over conventionallenses in that they can be molded flat and very thin. Because of theshape of the Fresnel lens, it can easily be integrated directly into thehousing of the luminometer, enhancing the light-tight propertiesnecessary for accurate imaging of low light samples. Furthermore,Fresnel lenses are much less expensive than comparable conventionalglass lenses.

As with any other lens, the total beam spread from a Fresnel lensdepends on the size of the source in relation to the focal length of thelens. Smaller sources, such as luminescent assay samples, and longerfocal lengths produce more compact beams. Since there are practicallimitations to minimizing the geometry and dimensions of the optics 80in the luminometer of the present invention, the use of Fresnel fieldlens 120 provides the greatest opportunity for fine-tuned optics. Theemissions from plate 10 pass through lens 120, and are refracted suchthat the image obtained at CCD 70 appears to look directly downward intoall wells, even laterally displaced (edge) ones. This feature istypically called “telecentric.”

Further in a preferred embodiment of the present invention, the filter130 may be configured on a wheel, wherein different filter elements mayoccupy different portions of the wheel, depending on the luminescentcharacteristics of the sample being analyzed. Filter 130 is preferablyinclined at an angle of 20°-30° relative to the CCD, so that strayreflected light is reflected outside the field of view. Specifically,the filter wheel 130 permits the selection of different wavelengthranges, which not only permit high quality imaging, but may be used toseparate the emissions of different reagents emitting at differentwavelengths. Again, the filter wheel 130 is controlled by centralprocessor 50, in coordination with central processor 50's control of theindividual sample wells 20 in the sample plate 10. In many assays, suchas those addressed in pending U.S. patent application Ser. No.08/579,787, incorporated by reference herein, multiple luminescentreagents, which emit at different wavelengths, are employed in a singlewell. Using multiple filters, each can be imaged in turn, and the trueconcentration can be calculated from the data set resulting usingpre-stored calibration factors. Filter 130 is preferably provided withan infrared (IR) filter operating in conjunction with the selectedbandpass, or as an independent element. Applicants have discovered thatstray IR radiation, resulting from the plate phosphorescence, resultingin abnormally high backgrounds. An IR filter suppresses this.

From the filter wheel 130, the sample-emitted light passes throughcamera lens 140, which in the preferred embodiment is a large aperture,low distortion, camera lens. Camera lens 140 focuses the image of thesample on the CCD chip 70. In the preferred embodiment of the presentinvention, CCD camera 70 is a cooled, low noise, high resolution device.The lense is preferably a 35 mm wide angle lens with a low light level(F1.4) large aperture character. Magnification of 3-6, preferably about5.5, is preferred. In preferred embodiments CCD camera 70 is providedwith an anti-blooming CCD chip, to enhance dynamic range, which is about10⁵ in the claimed invention, referred to as the NorthStar™ luminometer.Blooming occurs when a single pixel is overloaded with light and itsphotoelectrons overflow the CCD device well capacity, contaminatingsurrounding pixels. Further in the preferred embodiment of theluminometer of the present invention, the selected CCD camera includes aliquid cooled thermoelectric (Peltier) device providing cooling of theCCD to approximately −35° C., and the CCD has 1280×1024 pixels, each ofwhich are 16 μm square, producing a total active area of 20.5 mm×16.4mm. The quantum efficiency averages 15% over the range from 450nanometers to 800 nm. The output is digitized to 16 bit precision andpixels can be “binned” to reduce electronic noise.

By using the features disclosed herein, the luminometer of the presentinvention has a spatial resolution capable of providing high qualityimaging of high density sample trays. The noise performance and CCDtemperature are designed to provide the desired detection limit.

Mechanics

The mechanical systems of the luminometer workstation of this inventionare designed to achieve automated, high throughput precise delivery ofmicroplates in registration with a collimator 110 so as to be read bythe CCD Camera 70. To this end, as shown in FIG. 3, a cross-section ofthe inventive luminometer shuttle 200 translates from a load position202, where plates 10 are loaded on to the shuttle, preferably by arobotic device such as robot arm, and the shuttle 200 then translatestowards sample chamber 55, to read position 203. Shuttle 200 is causedto translate by a conventional stepper motor (not pictured). As shuttle200 advances toward sample chamber 55, it may stop underneath injector30. Injector 30 is more fully illustrated below in FIG. 4. Referringstill to FIG. 3, injector 30 delivers fluid reagents drawn fromreservoir 204. Syringe pump 205 draws the fluid reagents from reservoir204, and pumps the fluid to the injector tubes 40. Two way valve 206controls the passage of the fluid drawn by syringe pump 205 fromreservoir 204 and pumped by syringe pump 205 to the supply tubes 40. Inactual practice, there are as many injector tubes 40 as injection portsbeing used, and multiple syringe pumps 205 are also used. As will beshown below in FIG. 4, injector 30 has up to sixteen injection ports302. The plates used in conjunction with the luminometer when injectionis used are typically prepared with up to sixteen wells in a column. Asthe shuttle 200 advances plate 10 underneath injector 30, shuttle 200stops so that the first column 208 of wells is directly aligned underinjector 30. Precise amounts of analyte are delivered to the first setof wells, and shuttle 200 indexes forward one column, so as to injectreagent into the second column of wells 210. This process is repeateduntil all wells are filled. Thereafter, shuttle 200 advances forwardinto sample chamber 55 through hinged door 212. In the alternative, door212 may be a guillotine door or similar type of closing mechanism. Thewells of plate 10 are then read in sample chamber 55. Upon completion ofreading, shuttle 200 translates back to load position 202.

Before shuttle 200 advances to the injection bar, it may be necessary tofully prime the tube with fluid, so as to provide for precise deliveryinto the plate. Trough 304 swings out from its storage position parallelto the direction of travel of shuttle 200, shown by an arrow, to aposition directly underlying the injector 30, perpendicular to thedirection of travel. Fluid in the injector and tubes 204 are deliveredinto trough 304, and removed by suction. Trough 304 then returns to itsrest position, parallel to, and away from, the direction of travel ofthe shuttle 200, when the shuttle is moved toward the sample chamber 55.On its return trip to load position 202, locator 214 on shuttle 200 isengaged by cam 216. Locator 214 is mounted on a resilient means, suchthat when engaged by cam 216, the locator 214 recesses away from plate10. This permits removal of plate 10, and delivery from a robotic arm orother source of a fresh plate 10, without the requirement of preciselocation. As shuttle 200 moves away from load 202, locator 214 is urgedforward, firmly locating plate 10 in place. Plate 10 is held againstshoulder 217 by the resilient urging of locator 214.

It is important that each plate be precisely identified, so that resultsare correlated with the correct test samples. In most HTS laboratories,most microplates are labeled with a unique “bar code.” The label isoften placed on the surface perpendicular to the plane of the plateitself. To permit precise identification of each plate, a bar codereader 218 is mounted on the luminometer housing generally indicated at299 and directly above the door 212, for example on an arm or flange220. Bar code reader 218 is focused on a mirror 222 which in turnpermits reading directly off the front or leading edge of plate 10 as itapproaches on shuttle 200. Thus, before each plate arrives in the samplechamber, its identity has been precisely recorded in processor 50, andthe results obtained can be correlated therewith. Persons of ordinaryskill in the art will recognize that a variety of configurations ofalignment and placement of both bar code reader 218 and mirror 222 willresult in the desired identification.

As more clearly shown in FIG. 4, injector 30 may be precisely located byoperation of actuator wheel 306, provided with positions correspondingto the total number of wells on the plates being assayed. Similarly, thevertical position, to account for the different thicknesses of theplate, may be controlled by wheel 308. Given the simple translationmovement of shuttle 200, and the precise locating and identification ofeach plate carried, rapid cycling of micro-plate test plates into andout of sample chamber 55 can be effected.

As described above in connection with the optics system of theinvention, a filter is provided which includes or reflects passage oflight other than light falling within the selected wavelength of theluminescent emitter in use. The filter assembly is illustrated inexploded format in FIG. 5. Filter frame 502 is supported by arm 504which is connected to the hub of the filter wheel 506. Multipledifferent filters may be provided on a single wheel. The filter itself,508, is securely mounted on the frame and held there by cover 510, whichis secured to frame 502 by grommets, screws or other holding devices512. As noted, filter wheel is positioned so as to hold filter 508 inframe 502 at in incline with respect to collimator 110, of about 22°nominally, so as to direct any reflections outside the field of view.Light passes through the filter opening 514, in alignment with cameralens 140 and CCD camera 70. As further noted above, filter 508preferably includes an infrared block, either as a component of thefilter itself, or as a component provided in addition to the filter forthe measured light. An IR block is of value to prevent infraredemissions caused by extraneous radiation from altering the imagereceived by the CCD camera.

Optical chamber 60 is more fully illustrated in FIG. 6. As shown,optical chamber 60 is bounded by optical housing 602 in which fitssample housing 604. When a plate 10 is loaded into optical chamber 60,the plate is secured in sample housing 604 which is positioned inregistry with collimator 110, over which is provided Fresnel lens 120.While many luminescent assays can be provided at ambient temperatures,some require elevated temperatures. The luminometer of this device isprovided with a sample chamber in which the sample housing 604 carriesinsulation 606 which, in a preferred embodiment, is polyurethane foam,and heater element 608 to raise the temperature in the sample chamber 55above ambient temperature, up to about 42° C.

There is a tendency, even at ambient conditions, for condensation tocollect on the surface of the Fresnel lens 120, as a result of moisturecoming from the filled wells of plate 10. The defogger 610 directs astream of air heated just a few degrees, preferably about 2-3° degrees,above ambient conditions, or above the temperature of the chamber if thechamber is above ambient conditions, across the surface of the Fresnellens 120, effectively preventing condensation. Mounted at the top of theinterior of optical chamber 60 is filter motor 610 which drives filterwheel 612, on which may be mounted filters 614 of varying wavelength,for filtering undesirable wavelengths prior to imaging. Of course, aregion is provided, indicated at 616, in the optical housing 602 of theoptical chamber 60 for light to be directed onto the CCD camera afterpassing through the filter 614. The dimensions of optical chamber 60 areexaggerated in FIG. 6 to illustrate the relationship between the opticalchamber 60 and the filter wheel 612, and defogger 610. In practice, thefilter is located inside the optical chamber 60, and outside the samplehousing 604 but alternate locations are possible while still achievingthe desired function.

In FIG. 6A, a plan view of a novel robotic mechanism 616 is displayed ina preferred embodiment of the present invention, which provides capacityfor use in high throughput screening (HTS) applications. Referring toFIG. 6A, the operation is as follows: robot plate stacks 620, 622, 624,626, and 628 each can be filled with multiple sample plates 10, arrangedin a vertical stack. In the preferred embodiment of FIG. 6A, robot platestack 628 is designated as the discard stack. The remaining robot platestacks 620, 622, 624, and 626 can be programmed in order of delivery bysoftware controlled by processor 50 (not shown). In order to load orpick plates from any of these stacks, robot arm 630 moves vertically androtationally to the desired robot plate stack, under control of thesoftware programmed in processor 50.

When commanded by processor 50, transport 200 of the instrument willmove the sample plate 10 from load position 202 to the Read position203, and return it to load position 202 when imaging is complete. In theembodiment of the invention shown in FIG. 6A, the elapsed time betweenmoving the sample plate 10 from load position 202 to the read position203, and returning it to load position 202 is typically 30-120 seconds,including imaging time.

Staging positions 632 and 634 are located at 45 degree positionsrelative to the position of robot arm 630. In one embodiment, whileimaging is in process, the robot arm 630 can place a sample plate 10 atstaging position 632, in preparation for placing the sample plate 10 inload position 202. When the imaging is complete, the robot can move theread plate from load position 202 to staging position 634, then load theplate from staging position 632 to load position 202, and while thesample plate 10 is being imaged, the robot can move the plate fromstaging position 634 to the discard stack 628, and place a new sampleplate 10 at staging position 632. In practice, the staging positions areat approximately the same level as the load position, so movement isvery quick. In the preferred embodiment, the robot arm 630 can do thetime consuming moves to any of robot plate stacks 620, 622, 624, and 626while imaging is going on, rather than in series with imaging.

With the staging positions 632 and 634, the cycle time for a singlesample plate 10 is 2 moves from/to staging areas (3 seconds each), plus2 transport moves IN/OUT to read position 203 (3 seconds each), plus theintegration time (image exposure) time (typically 60 seconds), for atotal cycle time of 72 seconds. Without using staging positions 632 and634, the time would be 2 moves to stacks (30 seconds each), plus 2transports (3 seconds each), plus the integration time (typically 60seconds) for a total of 126 seconds. As described in the preferredembodiment of the robotic mechanism 616, the use of staging positions632 and 634 decreases cycle time by 43%.

Processing

As set forth above, the mechanical and optical systems of theluminometer workstation of the invention are designed to provideprecise, quantified luminescent values in an HTS environment, takingadvantage of the use of a Fresnel lens/collimator assembly to permitsingle image viewing by the CCD camera, and subsequent analysis. Thecollimator, the lens and the camera together combine to reducecross-talk experienced in prior art attempts. The signals obtained arefurther processed, as illustrated in FIG. 7, through software loadedonto processor 50, or other convenient method, to further refine thevalues obtained.

Prior to processing image data collected through the integratedmechanical and optical systems of the invention herein described, theintegrated processing component of the invention must first control themechanical alignment of those integrated mechanical and optical systemsfor reliable data collection. This process is conducted under control ofthe processor 50. To conduct an alignment test, the luminescencedetection of the present invention measures the light emitted from fourtest sample wells, called hot wells, of a test plate. In a preferredembodiment, the hot wells are located near each corner of the sampletray used for the alignment testing. The adjacent well crosstalk fromeach of the four hot wells is analyzed, and the values are compared.When the collimator is aligned precisely over the sample well tray, thecrosstalk values will be symmetrical for the four hot wells. Thesoftware of the present invention flags any errors detected, such asincorrect number of test sample wells, incorrect intensity, or incorrectlocation. After the detection of no errors or after the correction ofdetected and flagged errors, the software of the present inventionperforms a symmetry calculation to determine precise alignment of thesample well tray, collimator, Fresnel lens and CCD camera assembly. In aknown embodiment of the invention, known software techniques areemployed to perform the symmetry calculation process by performing thefollowing steps:

1. Extract the hot well and vertical and horizontal adjacent wellintensities;

2. Calculate the averages of the horizontal and vertical adjacent wellintensities separately for each hot well;

3. Calculate the differences between the actual adjacent intensity vs.the average for each of the horizontal and vertical directions;

4. Normalize the differences by the hot well intensity to convert to apercentage intensity value;

5. Find the worst case absolute value of the differences and displaythat as the overall misalignment;

6. Calculate the average X-direction (horizontal) misalignment byaveraging the four adjacent wells to the right (horizontal direction) ofthe hot wells;

7. Calculate the average Y-direction (vertical) misalignment byaveraging the four adjacent wells to the top (vertical direction) of thehot wells;

8. Calculate the rotational misalignment by averaging the left side hotwell vertical adjacent wells at the top of the hot wells, andsubtracting that from the average of the right side hot well verticaladjacent wells, thereby indicating any tilt in adjacent well values.

In order to clearly isolate and read each pixel, in step B, each imageis subjected to “masking”, a processing step whereby the edge of eachwell or corresponding light image is identified, or annotated, to setoff and clearly separate each well region of interest, as disclosed inU.S. patent application Ser. No. 09/351,660, incorporated herein byreference. Again, masking is performed for each of B₁, B₂ and B₃,referring to the pre-cursor, full integration time image and post-cursorimages, respectively. The images are then subjected to “outlier”correction, correcting or “shaving” outliers and anomalies. In thisprocess, the pixels within the region of interest are examined toidentify “outliers”—those that are in gross disagreement with theirneighbors, in terms of light intensity detected, and if the intensity ofa given pixel or small pixel area is significantly different thanneighboring pixels or pixel areas, then the average of the surroundingpixels or areas is used to replace erroneous data. This can be due torandom radiation, such as that caused by cosmic rays. In this process,this type of intensity is corrected.

Subsequently, in step C, each image C₁, C₂ and C₃ is subjected to darksubtraction, subtracting the dark background, so as to obtain averagepixel values within each mask-defined region of interest. Thesubtraction is done on a well-by-well basis from stored libraries whichare updated periodically.

Specifically, the dark subtraction is conducted to correct for the factthat even in the absence of light, CCD cameras can output low levelpixel or bin values. This value includes the electronic bias voltage,which is invariant of position and integration time, and the “darkcurrent,” which may vary by position, and is proportional to integrationtime and to the temperature of the CCD. The CCD may also have faultypixels that are always high level or saturated regardless of lightinput.

The processing software of the invention subtracts this background imageor data from the real sample well image data in step C. As persons ofordinary skill in the relevant art will recognize, it is known to take a“dark” image immediately before or after a real image, imaging for thesame integration time in both cases, and subtracting the “dark” imagedata from the real image data. In the preferred embodiment of theinvention, “dark” image data is collected intermittently, preferably atspecific time intervals. The initial “dark” image background data iscollected at startup, and then typically at four hour intervals duringimage processing operations.

Because the background image has an integration time-invariant componentand an integration time-variant component, data is collected for eachsample well at minimum integration time and at maximum integration time,and a “slope/intercept” line is calculated between the two data points,using known data analysis techniques. This calculation permits datainterpolation for any integration time between the minimum and maximum,and also permits data extrapolation for integration times below orbeyond the minimum and maximum integration times.

In a preferred embodiment of the invention, a CCD camera is employedthat has two separate “dark” current functions, caused by the CCD outputamplifier. Operation of the amplifier generates heat and necessarilycreates background “dark” image data. In the preferred embodiment, forintegration times of less than 10 seconds, the amplifier operatescontinuously, whereas for integration times of more 10 seconds, theamplifier remains off until immediately prior to the read operation. The“slope/intercept” line calculated for integration times of more than 10seconds will then necessarily have a lower slope than a“slope/intercept” line calculated for integration times of less than 10seconds. In step C, the processing software element allows separatecollection and least squares regression for both the 0 to 10 secondintegration time region and integration times that exceed 10 seconds.The “dark” background image data is stored separately for eachindividual AOI.

“Dark” current and bias can also vary over time. The processing softwareelement corrects for this effect by comparing the integration timenormalized (using the regression line technique described above) “darkreference” pixel values (outside the imaging field-described above),that were taken when the “dark” background images were taken, versus the“dark reference” pixel values taken while real sample well images arebeing taken. The difference between the values is then subtracted oradded, as applicable, as a global number, to the “dark” background data.This corrects for bias drift and also for global CCD temperature drift.

As mentioned, all of the above “dark background”interpolation/subtraction of step C is done on a well by well basis.

At step D, if pixel saturation has occurred such that the average of thepre-cursor and post-cursor image must be used, the image data ismultiplied by the reciprocal of the percentage represented by thepre-cursor images (e.g., 3%).

In step E, the well data is corrected for uniformity variations using acalibration file that is the reciprocal of the system response to aperfectly uniform input illumination.

In step F, the cross-talk correction is effected by processing the dataas a whole and preparing a final image in much the same fashion asreconstruction of three dimensional images from a two dimensional dataarray is practiced.

Specifically in a preferred embodiment of step F, the impulse responsefunction (IRF) is collected for all 96 wells of the 96 well plate type.This is done by filling one particular well in a given plate with a highintensity luminescent source, imaging the plate, and analyzing all ofthe wells in the plate for their response to the one high intensitywell. The IRF is collected for all of the wells individually byrepeating the process for every different well location desired for thecomplete data set. For 384 plate types, 96 sampling areas are selected,and data for the wells in between the selected sampled areas areinterpolated in two dimensions. In the preferred embodiment, the 96sampling areas comprise every second row and every second column,starting at the outside and working toward the center. Because in the384 well plates the number of rows and columns is even, the two centerrows and the two center columns are interpolated. The reflections in a384 well plate are also modeled, and used to predict and interpolatereflections for the missing input data. Further in the preferredembodiment, all wells are normalized to the well with the highestintensity.

Subsequently in step F, the two-dimensional array of well IRF values foreach well are “unfolded” into a one-dimensional column array, and thetwo-dimensional arrays of IRF values for other wells are added assubsequent columns, as shown in Chart 1 following:

Chart 1 - Unfolded Data Into Column 1 IRF for IRF for IRF for A1 B1 C1A1 A1 A1 Etc B1 B1 B1 C1 C1 C1 D1 D1 D1 E1 E1 E1 F1 F1 F1 G1 G1 G1 H1 H1H1 A2 A2 A2 B2 B2 B2 C2 C2 C2 Etc Etc Etc

The unfolded matrix, which has the form of an N×N matrix, where N=thenumber of wells to be corrected, comprises a full characterization ofthe instrument crosstalk, including reflection factors. This unfoldedmatrix is then inverted, using known matrix inversion techniques, andused as a correction to matrix multiply a one-dimensional matrixunfolded from real assay data. This arithmetic process may be shown asmatrix algebra:

[true source distribution]×[system IRF]=[instrument output]

solving for [true source distribution] produces

[true source distribution]={1/[system IRF]}×[instrument output]

Subsequently, the calculated well intensities resulting from the aboveprocessing are calibrated to an absolute parameter of interest, such asthe concentration of a known reporter enzyme. This calibration isconducted through a normalization process producing any of a variety ofcalibration curves, which will be familiar to those of ordinary skill inthe relevant art.

In optional step G, the processed image information is subjected to anynecessary post adjustment processing, for appropriate correlation withthe materials tested. Specifically, in a preferred embodiment, theprocessing software of the present invention is capable of performingmulti-component analysis. The basic problem is to calculate separatelythe concentration of a single reagent in a single sample containingother different reagents. Typically, the reagents used with theinvention are formulated so as to emit over different, but perhapsoverlapping, spectrums. As earlier described with respect to theintegrated optical element, the first step of separating the light frommultiple reagents is accomplished by optical bandpass filters, which aredesigned to maximize the sensitivity of the target reagent emission,while minimizing the sensitivity to other non-target reagent emission.In the present embodiment of the invention, there is one optical filterfor each target reagent emission spectrum.

Since optical filters are interference devices, their bandpasscharacteristics vary, dependent on the angle of incidence of theemission to be filtered. The angle of incidence will be unique for eachwell because each well's specific location is unique relative to theoptical filter. Accordingly, all calculations and filter coeficientsmust be unique per sample well. The multi-component calibration isperformed as follows:

Prior to the real multiplexed (multiple reagent) samples, standardscontaining only a single reagent in each well are imaged and analyzed.These standards will produce a set of coefficients to be usedcollectively as multi-component coefficients for each optical filter,for each well. For a given optical filter, the target reagent for thatfilter should produce the highest output. The other reagents may also.have spectra in the filter's bandpass, and will produce smaller outputs,which are a measure of the overlap of those nontarget reagent spectrainto the filter signal. For example, the filter's output for the targetreagent might be 850, and the filter's output for the other 2 reagentsmight be 100 and 50, respectively. If the 3 reagents were added togetherin a single well, the total output would be 1000, and the proportionswould be 850:100:50. These coefficients are measured for each welllocation and filter separately, which gives a complete set ofcoefficients for simultaneous equations. This will allow a solution forany combination of concentrations of reagent in one sample well. Furtherin the preferred embodiment, these coefficients will also be normalizedby the total intensity read in the “total emission” filter, so that thecalculation will result in the same intensity as the instrument wouldmeasure if only a single reagent was measured by the “total emission”filter. This calculation may be shown as follows for a simple case ofblue and green reagents (abbreviated as R in the calculations), and blueand green and total emission filters (abbreviated as F in thecalculations):

Let A=(output of the instrument for blue R thru the blue F)/(output ofinstrument for blue R thru total emission F);

Let B=(output of the instrument for green R thru the blue F)/(output ofinstrument for green R thru total emission F);

Let C=(output of the instrument for blue R thru the green F)/(output ofinstrument for blue R thru total emission F);

Let D=(output of the instrument for green R thru the green F)/(output ofinstrument for green R thru total emission F);

These coefficients are measured for each well prior to running amulti-color run. Then for a multi-reagent/color run,

(output of the instrument for the blue F)=A×(true intensity of blueR)+B×(intensity of green R);

and

(output of the instrument for the green F)=C×(true intensity of blueR)+D×(intensity of green R)

These 2 simultaneous equations are then solved for the true intensity ofthe blue and green reagents by the processing software, under control ofprocessor 50.

Further in step G, the raw output of the instrument for each filter isnormalized for integration time before solving the equations. Theresulting intensities could then be calibrated as concentration by useof standards as described in the previous section.

Finally, in step H, the analyzed data is presented in a user-acceptableformat, again controlled by processor 50.

The invention may be further understood by reference to examples ofassays practiced in HTS format, demonstrating the dynamic range andflexibility of the NorthStar™ luminometer.

EXAMPLES Example 1 Purified cAMP Quantitation

cAMP standards were serial diluted and added to a 96-well assay platewith alkaline phosphatase conjugated cAMP and anti-cAMP. Plates wereprocessed with the cAMPScreen™ protocol and imaged for 1 minute on theNorthStar™ 30 minutes after addition of CSPD®/Sapphire-II™. Asensitivity of 0.06 pM of purified cAMP is achieved with cAMP-Screen™ onthe NorthStar™ workstation. The results are depicted in FIG. 8.

Example 2 cAMP Induction in Adrenergic β2 Receptor-expressing C2 Cells

Adrenergic β2 Receptor-expressing C2 cells were plated in a 96-wellplate (10,000 cells/well) and stimulated with isoproterenol for 10minutes. cAMP production was quantitated in cell lysates using thecAMP-Screen™ assay. The assay plate was imaged for 1 minute on theNorthStar™, 30 minutes after addition of CSPD®/Sapphire-II™. IncreasingcAMP levels were detected on the NorthStar™ from the stimulatedadrenergic receptor. The results are depicted in FIG. 9.

Example 3 Luc-Screen™ Reporter Gene Assay in 96-, 384- and 1,536-wellFormat

pCRE-Luc-Transfected cells were seeded in 96-, 384- and 1,536-wellplates, incubated for 20 hours with forskolin, and assayed with theLuc-Screen™ system. PCRE-Luc contains the luciferase reporter gene underthe control of a cAMP response element (CRE). Forskolin inducesintracellular cAMP production through the irreversible activation ofadenylate cyclase. All plate formats demonstrate comparableforskolin-induced cAMP levels. The results are depicted in FIG. 10.

Example 4 Forskolin Induction of pCRE-Luc Transfected NIH-3T3 Cells

pCRE-Luc-Transfected cells were seeded in a 96-well plate. Four randomwells were induced for 17 hours with 1 mM forskolin and the entire platewas assayed with the Luc-Screen™ system. The results are shown in Figure11.

Example 5 Dual-Light® Quantitation of Luciferase & β-glactosidaseReporter Enzymes

NIH/3T3 cells were co-transfected with pCRE-Luc and pβgal-Control, andseeded into a 96-well microplate (2×10⁴ cells/well). Cells wereincubated with forskolin for 17 hours. Modified Dual-Light® Buffer A wasadded to cells and incubated for 10 minutes. Modified Dual Light® BufferB was injected and luciferase-catalyzed light emission was measuredimmediately. Thirty minutes later, Accelerator-II was added, and thenβ-galactosidase-catalyzed light emission was quantitated on theNorthStar™ HTS workstation. Quantitation is shown graphically in FIG.12.

Example 6 Normalized Fold Induction of Luciferase Reporter

Fold induction of luciferase activity was calculated followingnormalization to β-galactosidase activity. The Dual-Light® assay enablesthe use of a control reporter for normalization, or to monitornon-specific effects on gene expression. This is depicted in FIG. 13.

Example 7 Effect of BAPTA-AM on Antagonist Activity

CHO-Aeq-5HT2B cells were loaded with coelenterazine h+/−0.5 μM BAPTA-AMfor 4 hours. The antagonist methysergide was added to the charged cellsfor 30 minutes. 1 μM agonist a-Me-5HT was injected, and the emittedlight was integrated for 20 seconds on the NorthStar™ system. Thereported IC50 for methysergide (0.6 nM) is unchanged in the presence ofBAPTA-AM. The data obtained appears in FIG. 14.

Example 8 Effect of BAPTA-AM on Peptide Agonist Stimulated of the Orexin2 Receptor

CHO-Aeq-OX2-A2 cells (Euroscreen) were loaded with coelenterazineh+/−0.6 μM BAPTA-AM for 4 hours. The peptide agonist Orexin B wasinjected into the wells, and the emitted light was integrated for 20seconds on the NorthStar™. Using this assay on the NorthStar™ system,the reported EC50 for Orexin B (0.75 nM) is unchanged in the presence ofBAPTA-AM. This is shown in FIG. 15.

This invention has been described generically, by reference to specificembodiments and by example. Unless so indicated, no embodiment orexample is intended to be limiting. Alternatives will occur to those ofordinary skill in the art without the exercise of inventive skill, andwithin the scope of the claims set forth below.

What is claimed is:
 1. An imaging system, comprising: a charge coupleddevice; a collimator; a Fresnel lens positioned between the collimatorand the charge coupled device; and a camera lens positioned between theFresnel lens and the charge coupled device; wherein the collimator, theFresnel lens and the camera lens are arranged such that luminescentemissions from a source passing through the collimator toward the chargecoupled device are refracted by the Fresnel lens and focused by thecamera lens onto the charge coupled device to form an image of thesource.
 2. The imaging system of claim 1, further comprising at leastone optical bandpass filter positioned between the Fresnel lens and thecamera lens.
 3. The imaging system of claim 2, wherein the opticalbandpass filter is inclined at an angle with respect to the chargecoupled device.
 4. The imaging system of claim 3, wherein the opticalbandpass filter is inclined at an angle of 20° to 30° relative to thecharge coupled device.
 5. The imaging system of claim 1, furthercomprising an infrared filter positioned between the Fresnel lens andthe charge coupled device.
 6. A luminometer comprising: the imagingsystem of claim 1; an optical housing; a sample housing; a shuttleadapted to move a sample plate from a load position to a read positionin a sample housing; a central processor adapted to collect and analyzeimage data from the charge coupled device; wherein the charge coupleddevice and the camera lens are mounted outside of the optical housingand the collimator and the Fresnel lens are mounted inside of theoptical housing and wherein the optical housing comprises a region whichallows the passage of light such that luminescent emissions from asample plate in the read position can pass through the collimator andthe Fresnel lens and can be focused on the charge coupled device by thecamera lens.
 7. The luminometer of claim 6, further comprising anoptical bandpass filter mounted inside of the optical chamber betweenthe region which allows the passage of light and the Fresnel lens. 8.The luminometer of claim 7, wherein the optical bandpass filter is partof a filter assembly comprising a plurality of optical bypass filterelements.
 9. The luminometer of claim 8, wherein the filter assembly isa filter wheel comprising a hub and a plurality of radially extendingarms and wherein each of the plurality of optical bypass filter elementsare mounted to one of the arms.
 10. The luminometer of claim 9, furthercomprising a motor engaged with the hub of the filter wheel, wherein themotor is adapted to rotate the filter wheel to position any one of theplurality of filter elements between the charge coupled device and theFresnel lens.
 11. The luminometer of claim 6, further comprising adefogger adapted to direct a stream of heated air across a surface ofthe Fresnel lens.
 12. The luminometer of claim 6, further comprising aheater adapted to heat a sample mounted in the sample housing.
 13. Theluminometer of claim 6, further comprising an injector including aplurality of injector ports, wherein the injector is adapted to draw afluid from a reservoir and deliver the fluid through the injector portsto one or more sample wells on the sample plate.
 14. The luminometer ofclaim 13, further comprising one or more pumps adapted to draw the fluidfrom the reservoir and deliver the fluid from the reservoir to theinjector ports.
 15. The luminometer of claim 14, further comprising atwo-way valve, wherein the two way valve is adapted to control thepassage of fluid drawn from the reservoir and/or delivered to theinjector ports.
 16. The luminometer of claim 13, further comprising aplurality of injector tubes, each of the injector tubes adapted toconvey the fluid from the reservoir to an injector port.
 17. A method ofdetermining the mechanical misalignment of a sample tray in theluminometer of claim 6, wherein the sample tray comprises a plurality ofsample wells arranged in a grid of rows and columns, the methodcomprising: placing a luminescent composition in at least two hot wellsin a first column of the sample tray and in at least two hot wells in asecond column of the sample tray, wherein each of the hot wells arelocated near a corner of the grid and wherein each hot well issurrounded by four adjacent sample wells, two adjacent sample wells inthe same column and two adjacent sample wells in the same row, andwherein the four adjacent sample wells do not contain the luminescentcomposition; loading the sample tray onto the shuttle in the loadposition; moving the sample plate into the read position with theshuttle; imaging the sample tray with the charge coupled device;collecting the image data on the central processor; calculating theaverage misalignment of the sample tray in a first direction byaveraging the intensity attributed by the charge coupled device to thewells adjacent to each of the hot wells in the first direction, thefirst direction corresponding to a column or row of the sample tray;calculating the average misalignment of the sample tray in a seconddirection perpendicular to the first direction by averaging theintensity attributed by the charge coupled device to the wells adjacentto each of the hot wells in the second direction; calculating therotational misalignment of the sample tray by: averaging the intensityattributed by the charge coupled device to the adjacent wells in thesame column for each of the two hot wells in the first column; averagingthe intensity attributed by the charge coupled device to adjacent wellsin the same column for each of the two hot wells in the second column;and calculating the difference between the averages determined above,wherein the difference is a measure of the rotational misalignment. 18.The method of claim 17, further comprising determining the overallmisalignment of the sample tray in the luminometer by, for each of thehot wells: measuring the intensity of luminescent emissions attributedby the charge coupled device to the hot well; measuring the intensity ofluminescent emissions attributed by the charge coupled device to each ofthe two adjacent sample wells in the same column and to each of the twoadjacent sample wells in the same rows; calculating an average for theintensity values for the two adjacent wells in the same column and anaverage for the intensity values for the two adjacent wells in the samerow; calculating the difference between the actual intensity attributedby the charge coupled device to each of the two adjacent wells in thesame column to the average intensity for the two adjacent wells in thesame column; calculating the difference between the actual intensityattributed by the charge coupled device to each of the two adjacentwells in the same row to the average intensity for the two adjacentwells in the same row; normalizing the differences by dividing thedifferences by the respective hot well intensity and, optionally,converting the result to a percentage; and taking the absolute value ofthe normalized differences; wherein the largest absolute value is ameasure of the overall misalignment of the sample tray.
 19. The methodof claim 17, further comprising adjusting the read position of theshuttle to the luminometer to reduce the rotational misalignment and/orthe average misalignment of the sample tray in the first and seconddirections of the sample trays.
 20. A method for high throughputscreening of a plurality of sample trays in the luminometer of claim 6,wherein each of the sample trays comprises a plurality of luminescentsamples, the method comprising screening a plurality of sample trays insuccession, each sample comprising a plurality of samples, wherein, foreach sample tray, the method further comprises: loading the sample trayonto the shuttle in the load position; moving the sample plate into theread position with the shuttle; taking at least one image of the sampletray with the charge coupled device at an integration time; collectingthe image data generated by the charge coupled device on the centralprocessor; processing the image data; and removing the sample tray fromthe luminometer.
 21. The method of claim 20, wherein the step of takingat least one image comprises: taking a first precursor image of thesample tray with the charge coupled device at a first integration time;taking a second full integration time image of the sample tray with thecharge coupled device at a second integration time greater than thefirst integration time; and taking a third post cursor image of thesample tray with the charge coupled device at a third integration timeless than the second integration time; analyzing the full integrationtime image data with the central processor to determine if more than sixpixels are saturated; if more than six pixels of the full integrationtime image are saturated, using the full integration time normalizedaverage of the pre and post cursor image data for subsequent processing;and if six or fewer pixels are saturated, using the full integrationtime image data as the image data for subsequent processing.
 22. Themethod of claim 20, wherein the step of processing the image data on thecentral processor comprises a process selected from the group consistingof: subjecting the image data to masking to extract the data for eachparticular well; subjecting the image data to outlier shaving;subjecting the image data to dark subtraction; correcting the image datafor uniformity variations in the charge coupled device and opticalsystem; correcting the image data for cross-talk from adjacent wells;calibrating the image data to a parameter of interest; subjecting theimage data to post adjustment processing; and combinations thereof. 23.The method of claim 22, wherein the step of outlier shaving comprises:identifying outliers by comparing the light intensity for a pixel orpixel area to the light intensity for neighboring pixels or pixel areas,wherein outliers have a light intensity that is significantly differentthan the neighboring pixels or pixel areas; and for each outlieridentified, calculating the average intensity value for the neighboringpixel or pixel areas of each outlier and substituting the average valueof intensity of the neighboring pixel or pixel areas for the intensitydata of the outlier.
 24. The method of claim 22, wherein the step ofsubjecting the image data to dark subtraction comprises: collecting.dark image data for the luminometer at a first integration time and asecond integration time before imaging a sample plate; calculating aslope/intercept line for the two points using least squares regression;normalizing the dark image data for the integration time of the imageusing the slope/intercept line; and subtracting the integration timenormalized dark image data from the image data.
 25. The method of claim24, wherein dark image data is collected before imaging the first sampleplate and periodically during high throughput screening, the methodfurther comprising subtracting the integration time normalized darkimage data calculated from the most recently collected dark image datafrom the image data.
 26. The method of claim 22, wherein the step ofcorrecting the image data for cross-talk comprises: for each sample wellon a sample plate, filling the sample well with a luminescent compound,imaging the sample plate, collecting the intensity data for the sampleplate, forming a two dimensional array from the intensity data, andunfolding the two-dimensional array into a one dimensional column array;forming a two-dimensional array from the column arrays for each samplewell on the sample plate; mathematically inverting the two-dimensionalarray; unfolding the image data to form a one dimensional matrix; andmultiplying the unfolded image data by the inverted two-dimensionalarray.
 27. The method of claim 22, wherein the step of correcting theimage data for cross-talk comprises: for each sample well in a first setof selected sample wells on a sample plate, filling the sample well witha luminescent compound, imaging the sample plate; collecting theintensity data for the sample plate; forming a two dimensional arrayfrom the intensity values and unfolding the two-dimensional array into aone dimensional column array; for each of the remaining sample wells onthe sample plate, interpolating intensity data from the intensity datacollected for the first set of selected sample wells, forming a twodimensional array from the interpolated intensity values and unfoldingthe two-dimensional array into a one dimensional column array; forming atwo-dimensional array from the column arrays for each sample well;inverting the two-dimensional array; unfolding the image data to form aone dimensional matrix; and multiplying the unfolded image data by theinverted two-dimensional array.
 28. The method of claim 27, wherein thesample tray comprises 384 sample wells arranged in 16 rows and 24columns, the method further comprising collecting intensity data for the96 sample wells in rows 1, 3, 5, 7, 10, 12, 14, 16 and in columns 1, 3,5, 7, 9, 11, 14, 16, 18, 20, 22, 24 and interpolating the intensity datafor the remaining wells.
 29. The method of claim 20, wherein theplurality of luminescent samples each comprise more than one luminescentreagent, each reagent adapted to emit luminescent emissions over adifferent wavelength spectrum, the method further comprising, for eachreagent: collecting image data using an optical bandpass filter, whereinthe optical bandpass filter is adapted to maximize the sensitivity ofthe charge coupled device to emissions from the reagent; and determiningthe contribution to the image data of the luminescent emissions fromeach of the reagents.
 30. The method of claim 29, wherein the step ofdetermining the contribution of the luminescent emissions from each ofthe reagents comprises: a) for each combination of luminescent reagentand optical bandpass filter, filling each sample well on a sample platewith a sample comprising the luminescent reagent as the sole luminescentreagent, imaging the sample plate using the optical bandpass filter andthe total emission filter as a reference, and collecting the image data;b) normalizing the bandpass filter intensity by the total emissionfilter intensity; c) generating a set of simultaneous equations for theimage data for the multi-reagent samples as measured through each filteras a function of the contribution to the image data of each of theindividual reagents, wherein the image data collected in (a) andnormalized in (b) for each combination of luminescent reagent and filterform the respective coefficients for the contribution of each of theluminescent reagents; and c) solving the set of simultaneous equationsfor the contribution of each of the reagents.